Bacterial conjugated linoleic acid bio-fortification of synbiotic yogurts using Propionibacterium freudenreichii as adjunct culture

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Omid Zahed
Kianoush Khosravi-Darani
Amir Mohammad Mortazavian
Abdorreza Mohammadi


conjugated linoleic acid, probiotic, Propionibacterium freudenreichii, yogurt


In this study, Propionibacterium freudenreichii was used for in situ production of conjugated linoleic acid (CLA) in yogurt. Firstly, effects of process variables, including strain type, percentage of milk fat, percentage of inoculum, quantity of sunflower oil, concentration of inulin, temperature of fermentation and time of storage at 4°C, on production of CLA by Propionibacterium freudenreichii were investigated using screening method of the Plackett–Burman design. Then optimisation of CLA production process was conducted using three major factors of milk fat percentage, inulin concentration and storage time at 4°C using central composite design. Analysis of variance established that the models were highly significant (P ≤ 0.05). The model demonstrated that the production of CLA was affected by these three factors. Optimised CLA production by Propionibacterium freudenreichii ssp. shermanii in yogurts was achieved after 17 days of storage at 4°C in skim-milk containing 1.75% (w/w) fat and 2.25% (w/v) inulin as prebiotic. Reconfirmation test established that at the highlighted optimum conditions, the highest concentration of produced CLA was 6.4 mg g–1 lipid in yogurt, which is a 256% increase in total CLA production, compared with control samples. Results demonstrated that Propionibacterium freudenreichii ssp. shermanii not only leads to production of synbiotic yogurts containing inulin but also increases CLA production in yogurts.

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Abou Ayana I.A., El-Deeb A.M. and Ibrahim A.E. 2016. Research article using of dairy propionibacteria as bio-preservative in Kareish cheese. International Journal of Dairy Science 11: 114–123. 10.3923/ijds.2016.114.123

Ahmadi N., Khosravi-Darani K., Zarean-Shahraki S., Mortazavian M. and Mashayekh S. 2015. Fed-batch fermentation for propionic, acetic and lactic acid production. Oriental Journal of Chemistry 31: 581–590. 10.13005/ojc/310174

Akalın A., Tokuşoğlu Ö., Gönç S. and Aycan Ş. 2007. Occurrence of conjugated linoleic acid in probiotic yoghurts supplemented with fructooligosaccharide. Intnational Dairy Journal 17: 1089–1095. 10.1016/j.idairyj.2007.02.005

Alonso L., Cuesta E. and Gilliland S. 2003. Production of free conjugated linoleic acid by Lactobacillus acidophilus and Lactobacillus casei of human intestinal origin. International Journal of Dairy Science 86: 1941–1946. 10.3168/jds.S0022-0302(03)73781-3

Balci Yuce H., Akbulut N., Ocakli S., Kayir O. and Elmastas M. 2017. The effect of commercial conjugated linoleic acid products on experimental periodontitis and diabetes mellitus in Wistar rats. Acta Odontologica Scandinavica 75: 21–29. 10.1080/00016357.2016.1244355

Boylston T. and Beitz D. 2002. Conjugated linoleic acid and fatty acid composition of yogurt produced from milk of cows fed soy oil and conjugated linoleic acid. Journal of food science 67: 1973–1978. 10.1111/j.1365-2621.2002.tb08755.x

Capela P., Hay T. and Shah N.P. 2006. Effect of cryoprotectants, prebiotics and microencapsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt. International Food Research Journal 39: 203–211. 10.1016/j.foodres.2005.07.007

Corbo M.R., Bevilacqua A., Petruzzi L., Casanova F.P. and Sinigaglia M. 2014. Functional beverages: the emerging side of functional foods: commercial trends, research, and health implications. Comprehensive Reviews in Food Science and Food Safety 13: 1192–1206. 10.1111/1541-4337.12109

Cousin F.J., Jouan-Lanhouet S., Théret N., Brenner C., Jouan E., Le Moigne-Muller G., Dimanche-Boitrel M.-T. and Jan G. 2016. The probiotic propionibacterium freudenreichii as a new adjuvant for TRAIL-based therapy in colorectal cancer. Oncotarget 7: 7161. 10.18632/oncotarget.6881

Dave R. and Shah N. 1996. Evaluation of media for selective enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, and bifidobacteria. International Journal of Dairy Science 79: 1529–1536. 10.3168/jds.S0022-0302(96)76513-X

Effat B.A., Ibrahim M.K., Tawfik N.F., Mehanna N.S. and Soliman N.R. 2019. Effect of different prebiotics on survival and viability of some dairy propionibacteria. International Journal of Advanced Research in Biological Sciences 6: 99–104. 10.22192/ijarbs.2019.06.08.014

Ekinci F. and Gurel M. 2008. Effect of using propionic acid bacteria as an adjunct culture in yogurt production. International Journal of Dairy Science 91: 892–899. 10.3168/jds.2007-0244

Farhadi S., Khosravi-Darani K., Mashayekh M., Mortazavian A., Mohammadi A. and Shahraz F. 2012. Effect of incubation temperature and inoculation ratio of starter culture on propionic acid production in dairy beverage fermented with propionibacterium. Iranian Journal of Nutritional Sciences 7: 41–50.

Fukuda S., Suzuki Y., Murai M., Asanuma N. and Hino T. 2006. Isolation of a novel strain of Butyrivibriofibrisolvens that isomerizes linoleic acid to conjugated linoleic acid without hydrogenation, and its utilization as a probiotic for animals. Journal of Applied Microbiology 100: 787–794. 10.1111/j.1365-2672.2006.02864.x

Gorret N., Maubois J., Ghoul M. and Engasser J. 2001. Exopolysaccharide production by Propionibacterium acidipropionici on milk microfiltrate. Journal of Applied Microbiology 90: 779–787. 10.1046/j.1365-2672.2001.01306.x

Grinstead D. and Barefoot S. 1992. Jenseniin G, a heat-stable bacteriocin produced by propionibacterium jensenii P126. Applied and Environmental Microbiology 58: 215–220. 10.1128/AEM.58.1.215-220.1992

Grunert K.G. 2005. Food quality and safety: consumer perception and demand. European Review of Agricultural Economics 32: 369–391. 10.1093/eurrag/jbi011

Güler-Akın M.B. and Akın M.S. 2007. Effects of cysteine and different incubation temperatures on the microflora, chemical composition and sensory characteristics of bio-yogurt made from goat’s milk. Food Chemistry 100: 788–793. 10.1016/j.foodchem.2005.10.038

Ha Y.L., Grimm N.K. and Pariza M.W. 1989. Newly recognized anticarcinogenic fatty acids: identification and quantification in natural and processed cheeses. Journal of Agricultural and Food Chemistry 37: 75–81. 10.1021/jf00085a018

Hennessy A.A., Barrett E., Ross R.P., Fitzgerald G.F., Devery R. and Stanton C. 2012. The production of conjugated α-linolenic, γ-linolenic and stearidonic acids by strains of bifidobacteria and propionibacteria. Lipids 47: 313–327. 10.1007/s11745-011-3636-z

Hernandez E. 2013. Enrichment of baked goods with omega-3 fatty acids. In: “Food enrichment with omega-3 fatty acids”. C. Jacobsen, N. S. Nielsen, A. Frisenfeldt Horn and A-D. Moltke Sorensen (Ed), Elsevier, pp. 319–335. Woodhead Publishing, Cambridge. 10.1533/9780857098863.3.319

Holzapfel W.H. and Schillinger U. 2002. Introduction to pre-and probiotics. International Food Research Journal. 35: 109–116. 10.1016/S0963-9969(01)00171-5

Khan M.M., Mir N.A. and Khan M.M. 2011. Production of vitamin B12 by improved strains of propionibacterium freudenreichii. Biotechnology & Bioengineering 1: 19–24. 10.1016/j.ejbt.2016.07.004

Khodaiyan F., Razavi S.H. and Mousavi S.M. 2008. Optimization of canthaxanthin production by Dietzianatronolimnaea HS-1 from cheese whey using statistical experimental methods. Biochemical Engineering Journal 40: 415–422. 10.1016/j.bej.2008.01.016

Khosravi-Darani K. and Zoghi A. 2008. Comparison of pretreatment strategies of sugarcane baggase: experimental design for citric acid production. Bioresource Technology 99: 6986–6993. 10.1016/j.biortech.2008.01.024

Kim Y.J. 2003. Partial inhibition of biohydrogenation of linoleic acid can increase the conjugated linoleic acid production of butyrivibrio fibrisolvens A38. Journal of Agricultural and Food Chemistry 51: 4258–4262. 10.1021/jf034057r

Kim Y.J., Liu R.H., Bond D.R. and Russell J.B. 2000. Effect of linoleic acid concentration on conjugated linoleic acid production by butyrivibrio fibrisolvens A38. Applied and Environmental Microbiology 66: 5226–5230. 10.1128/AEM.66.12.5226-5230.2000

Kishino S., Ogawa J., Omura Y., Matsumura K. and Shimizu S. 2002. Conjugated linoleic acid production from linoleic acid by lactic acid bacteria. Journal of the American Oil Chemists’ Society 79: 159-163. 10.1007/s11746-002-0451-4

Kouya T., Tobita K., Horiuchi M., Nakayama E., Deguchi H., Tanaka T. and Taniguchi M. 2008. Production of extracellular bifidogenic growth stimulator (BGS) from propionibacterium shermanii using a bioreactor system with a microfiltration module and an on-line controller for lactic acid concentration. Journal of Bioscience and Bioengineering 105: 184–191. 10.1263/jbb.105.184

Lin T.Y. 2000. Conjugated linoleic acid concentration as affected by lactic cultures and additives. Food Chemistry 69: 27–31. 10.1016/S0308-8146(99)00218-6

Lin T.Y., Lin C.-W. and Lee C.-H. 1999. Conjugated linoleic acid concentration as affected by lactic cultures and added linoleic acid. Food Chemistry 67: 1–5. 10.1016/S0308-8146(99)00077-1

Masso-Welch P.A., Zangani D., Ip C., Vaughan M.M., Shoemaker S.F., McGee S.O. and Ip M.M. 2004. Isomers of conjugated linoleic acid differ in their effects on angiogenesis and survival of mouse mammary adipose vasculature. Journal of Nutrition 134: 299–307. 10.1093/jn/134.2.299

Mohanty D., Misra S., Mohapatra S. and Sahu PS. 2018. Prebiotics and synbiotics: Recent concepts in nutrition. Food bioscience 26: 152-160. 10.1016/j.fbio.2018.10.008

Nieman C. 1954. Influence of trace amounts of fatty acids on the growth of microorganisms. Bacteriological Reviews 18: 147.

Ogawa J., Matsumura K., Kishino S., Omura Y. and Shimizu S. 2001. Conjugated linoleic acid accumulation via 10-hydroxy-12-octadecaenoic acid during microaerobic transformation of linoleic acid by Lactobacillus acidophilus. Applied and Environmental Microbiology 67: 1246–1252. 10.1128/AEM.67.3.1246-1252.2001

Oliveira R.P., Florence A.C., Silva R.C., Perego P., Converti A., Gioielli L.A. and Oliveira M.N. 2009. Effect of different prebiotics on the fermentation kinetics, probiotic survival and fatty acids profiles in nonfat symbiotic fermented milk. International Journal of Food Microbiology 128: 467–472. 10.1016/j.ijfoodmicro.2008.10.012

Olson J.M., Haas A.W., Lor J., McKee H.S. and Cook M.E. 2017. A comparison of the anti-inflammatory effects of cis-9, trans-11 conjugated linoleic acid to celecoxib in the collagen-induced arthritis model. Lipids. 52: 151–159. 10.1007/s11745-016-4228-8

Pandey S.M. and Mishra H. 2015. Optimization of the prebiotic & probiotic concentration and incubation temperature for the preparation of synbiotic soy yoghurt using response surface methodology. Food Science 62: 458–467. 10.1016/j.lwt.2014.12.003

Rad A.H., Khosroushahi A.Y., Khalili M. and Jafarzadeh S. 2016. Folate bio-fortification of yoghurt and fermented milk: a review. Dairy Science & Technology 96: 427–441. 10.1007/s13594-016-0286-1

Ranadheera C.S., Evans C., Adams M. and Baines S. 2012. Probiotic viability and physico-chemical and sensory properties of plain and stirred fruit yogurts made from goat’s milk. Food Chemistry 135: 1411–1418. 10.1016/j.foodchem.2012.06.025

Ross R., Mills S., Hill C., Fitzgerald G. and Stanton C. 2010. Specific metabolite production by gut microbiota as a basis for probiotic function. International Dairy Journal 20: 269–276. 10.1016/j.idairyj.2009.12.003

Salem MM., El-Gawad A., Hassan FA. and Effat BA. 2007. Use of synbiotics for production of functional low fat Labneh. Polish Journal of Food and Nutrition Sciences 57: 151-159.

Shantha NC., RAM LN., O’leary J., HICKS CL. and DECKER EA. 1995. Conjugated linoleic acid concentrations in dairy products as affected by processing and storage. Journal of Food Science 60: 695-697. 10.1111/j.1365-2621.1995.tb06208.x

Tharmaraj N. and Shah N. 2003. Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and propionibacteria. Journal of Dairy Science 86: 2288–2296. 10.3168/jds.S0022-0302(03)73821-1

Van Wyk J., Morkel R.A. and Dolley L. 2018. Metabolites of propionibacterium: techno-and biofunctional ingredients. Alternative and Replacement Foods. Elsevier, pp. 205–260. 10.1016/B978-0-12-811446-9.00008-3

Wang L.-M., Lv J.-P., Chu Z.-Q., Cui Y.-Y. and Ren X.-H. 2007. Production of conjugated linoleic acid by propionibacterium freudenreichii. Food Chemistry 103: 313–318. 10.1016/j.foodchem.2006.07.065

Xu S., Boylston T.D. and Glatz B.A. 2004. Effect of lipid source on probiotic bacteria and conjugated linoleic acid formation in milk model systems. Journal of the American Oil Chemists’ Society 81: 589–595. 10.1007/s11746-006-0946-z

Xu S., Boylston T.D. and Glatz B.A. 2005. Conjugated linoleic acid content and organoleptic attributes of fermented milk products produced with probiotic bacteria. Journal of Agricultural Food Chemistry 53: 53(23): 9064–9072. 10.1021/jf051030u

Yang B., Gao H., Stanton C., Ross R.P., Zhang H., Chen Y.Q., Chen H. and Chen W. 2017. Bacterial conjugated linoleic acid production and their applications. Progress in Lipid Research 68: 26–36. 10.1016/j.plipres.2017.09.002

Zárate G 2012. Dairy propionibacteria: less conventional probiotics to improve the human and animal health. In: “Probiotic in Animals”. Rigobelo E (Ed), pp 153–202. InTech, Rijeka. 10.5772/50320

Zárate G., Babot J., Argañaraz-Martinez E., Lorenzo-Pisarello M. and Perez Chaia A. 2011. Dairy propionibacteria: technological importance and probiotic potential for application on human and animal nutrition. In: Filip Rosana, editor. Paper presented at the Multidisciplinary Approaches on Food Science and Nutrition for the XXI Century Research Signpost. Trivandrum-695-023, Kerala, India: Transworld Research Network India, pp. 75–213.